【作者】 王斌；

【导师】 伍国耀； 戴兆来；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2018， 博士

【摘要】 本研究主要探讨了日粮添加L-色氨酸(L-Tryptophan,Trp)对断奶仔猪肠道炎症、屏障功能和免疫功能的影响,并通过小鼠和大鼠实验性肠道炎症模型研究可能的分子机制。试验一旨在研究日粮添加色氨酸对乙酸(Acetate)诱导的断奶仔猪肠道炎症的影响。试验选取54头21日龄大×长二元断奶去势公猪(平均体重为6.92 ±0.04 kg),根据体重随机分为6组:0%Trp、0%Trp+Acetate、0.2%Trp、0.2%Trp+Acetate、0.4%Trp 和 0.4%Trp+Acetate;每组 9头猪。试验日粮在基础日粮上添加0%、0.2%或0.4%色氨酸,各组日粮均等能等氮。适应期3d,试验期12 d。在试验d 0-7各组仔猪自由采食。在试验d 8灌注10 mL 10%乙酸诱导肠道炎症或灌注等体积0.9%生理盐水作为对照,试验d 8-12以乙酸处理组断奶仔猪采食量为标准进行配对饲喂。结果表明,0.2%色氨酸提高了断奶仔猪d0-7平均日增重和饲料利用效率(P<0.05);0.2%和0.4%色氨酸缓解了乙酸引起的断奶仔猪d 8-12平均日增重和饲料利用效率的降低(P<0.05)、结肠组织结构的破坏、紧密连接蛋白ZO-1表达的下调(P<0.05)、炎症因子(IL-1β、IL-6和IL-8)和Toll样受体TLT4 mRNA表达的上调(P<0.05)、炎症(STAT3和p65)和凋亡相关蛋白(Cleaved-Caspase-3)表达的上调(P<0.05)、5-羟色胺降解转运载体SERT mRNA表达的下调(P<0.05)。因此日粮添加色氨酸可以提高断奶仔猪的生长性能,并且可能通过调控5-羟色胺通路缓解乙酸诱导的断奶仔猪肠道屏障功能紊乱和炎症反应。试验二旨在研究色氨酸对葡聚糖硫酸钠(Dextransodium sulfate,DSS)诱导的小鼠肠道炎症的缓解作用和机制。试验选取80只8周龄C57BL/6J雄性小鼠(平均体重为19.9±0.22g),根据体重随机分为4个组:对照、DSS、Trp和Trp+DSS;每组5个重复,每个重复4只小鼠。适应期7d,试验期17d。试验期间饮水中色氨酸的添加量为0或0.1mg/gBW/d;试验d8-14使用2%DSS(w/v)建立小鼠急性结肠炎模型;试验d 15-17所有DSS处理小鼠恢复正常饮水。结果表明,色氨酸提高了试验d 0-7小鼠的平均日采食量和平均日饮水量(P<0.05);色氨酸缓解了DSS诱导的小鼠体重的下降、死亡率的增加、结肠的缩短、疾病活性指数的上升和肠道病理损伤(P<0.05);色氨酸改善DSS诱导的结肠屏障损伤和炎症反应主要是通过维持结肠紧密连接蛋白(ZO-1、Occludin 和 Claudin-1)结构和表达(P<0.05),抑制黏蛋白(MUC1 和 MUC2)mRNA表达的提高(P<0.05),减少免疫细胞(中性粒细胞、巨噬细胞和T细胞)向结肠固有层和黏膜下层的浸润,抑制促炎性细胞因子IL-6、TNF-α、IL-17a、G-CSF和MIP-1amRNA表达的升高(P<0.05),促进抑炎性细胞因子TGF-β2和TGF-β3 mRNA的表达(P<0.05),抑制TLR2和TLR4 mRNA以及NF-κB蛋白表达的上调(P<0.05)。色氨酸同样缓解了 DSS诱导的空肠肠道屏障损伤和炎症反应。这可能与色氨酸维持了结肠和空肠5-羟色胺稳态(分别缓解结肠SERT和空肠TPH1基因表达的下调)以及调节5-羟色胺受体的基因表达(均促进HTR1和HTR4基因表达)相关(P<0.05)。因此饮水添加色氨酸缓解DSS引起的小鼠体重下降和死亡率增加,并且可能通过调控5-羟色胺信号通路缓解DSS诱导的小鼠肠道屏障功能紊乱和炎症反应。试验三旨在研究色氨酸添加对脂多糖诱导大鼠空肠葡萄糖和氨基酸转运能力的影响。试验选取24只7周龄SD大鼠(平均体重191±7.2 g),根据体重随机分为3个组(对照、脂多糖、色氨酸+脂多糖组),每组8只大鼠。适应期7 d,试验期9 d。试验期间饮水中色氨酸的添加量为0或0.1mg/gBW/d;在试验d8大鼠腹腔注射脂多糖(5mg/kgBW)诱导肠道炎症或注射相同体积的0.9%生理盐水作为对照处理。注射24 h后采样,通过Ussing Chamber技术检测空肠肠道通透性、葡萄糖和氨基酸转运能力以及转运载体基因表达。结果表明,饮水中添加色氨酸缓解了脂多糖引起大鼠空肠通透性的增加(P<0.05)、葡萄糖和氨基酸(谷氨酸、精氨酸、谷氨酰胺、色氨酸和亮氨酸)钠离子转运的降低(P<0.05)以及葡萄糖(SGLT1)、氨基酸(SNAT2和LAT2)转运载体和Na+/K+-ATP酶(ATP1A2)mRNA表达的降低(P<0.05)。综上所述,色氨酸可能通过维持肠道紧密连接蛋白表达和分布以及黏蛋白基因表达、调控促炎性和抑炎性细胞因子的基因表达、抑制炎症信号通路的激活以及促进肠道葡萄糖和氨基酸转运缓解试验性肠道炎症引起的肠道屏障损伤和炎症反应。色氨酸缓解实验性肠道炎症引起的5-羟色胺含量和受体表达的改变,提示5-羟色胺可能参与色氨酸修复肠道屏障和缓解肠道炎症的作用。本研究为色氨酸在动物和人类肠道健康中的应用提供了试验依据。更多还原

【Abstract】 This study was conducted to determine the regulatory effect of L-tryptophan(Trp)on the intestinal inflammation,mucosal barrier function,immunemodulation and potential underlying mechanisms in animals.Experiment 1 The objective was to determine the effects of dietary supplementation with Trp on acetate-induced intestinal inflammation in weaned piglets.Fifty-four 21 day-weaned crossbred castrated male piglets(6.92±0.04 kg body weight(BW))were randomly allotted into one of six groups(3 Trp levels × 2 acetate levels):0%Trp,0%Trp+acetate,0.2%Trp,0.2%Trp+acetate,0.4%Trp,and 0.4%Trp+acetate;9 piglets per group.Piglets were fed a basal diet supplemented with 0%,0.2%or 0.4%of Trp throughout the 12 d-period of trail after a 3 d-period of adaptation.During d 0-7 of the trail,piglets in each group had free access to the diet as indicated.On d 8 of the trail,piglets were intrarectally administrated with 10 mL of 10%acetate or 0.9%saline served as control.During d 8-12 of the trail,piglets of other 5 groups were pair-fed the same amount of feed per kg BW as piglets in the acetate group.Compared with control piglets,dietary supplementation with 0.2%Trp increased daily weight gain and effeciency of feed utilization during d 0-7 of the trail(P<0.05);dietary supplementation with 0.2%and 0.4%Trp restored acetate-induced adverse changes,including reductions in daily weight gain and efficiency of feed utilization during d 8-12 of the trail(P<0.05).Acetate-induced colonic structural damage,down-regulation of tight junction protein ZO-1(P<0.05),up-regulation of inflammatory mediators(IL-lβ,IL-8 and TLR4)(P<0.05),activation of STAT3,NF-κB and Cleaved-Caspase-3(P<0.05),as well as down-regulation of gene expression of serotonin(5-HT)re-uptake transporter(SERT)were partially reversed by Trp(P<0.05).Therefore,dietary supplementation of Trp attenuated acetate-induced intestinal inflammation was associated with 5-HT signaling.Experiment 2 The objective was to determine the potential underlying mechanim of Trp in alleviating dextran sodium sulfate(DSS)-induced intestinal inflammation in mice.A Total of 40 eight-week old male C57BL/6J mice(19.9±0.22 g BW)were randomly divided into four groups(2 Trp levels x 2 DSS levels):control,DSS,Trp,and Trp+DSS;5 replicates,and 2 mice per replicate.Mice were supplemented with 0 or 0.1 mg Trp/g BW/d in water for 17 d.DSS(2%w/v)was orally administrated on d 8-15.Results indicated that Trp attenuated the severity of DSS colitis by increasing feed and water intake,abolished partially the body weight loss,mortality rate,colon shortening,disease actitvity index and colonic morphology(P<0.05).Trp alleviated DSS-induced intestinal barrier dysfunction and inflammation by regulating tight junction protein(ZO-1,Occludin and Claudin-1)and gene expression of mucin(MUC1 and MUC2)(P<0.05),inhibiting infiltration of inflammatory cells and sepressing pro-inflammaoty mediators(IL-6,TNF-a,IL-17a,G-CSF and MIP-1a)(P<0.05),and enhancing anti-inflammaoty cytokines(TGF-β2 and TGF-β3)(P<0.05).DSS-induced activation of inflammatory signaling(TLR2,TLR4 and NF-κB)was reversed by Trp(P<0.05).Trp also prevented DSS-induced intestinal damage and inflammation in jejunum.This function of Trp was related to the maintenance of 5-HT content(the inhibition of down-regulation of SERT and TPH1 in colon and jejunum,respectively)(P<0.05),and the regulation of 5-HT receptors(up-regulation of HTR1A and HTR4 both in colon and jejunum)(P<0.05).Therefore,Trp alleviates DSS-induced body weigh loss and mortality rate in mice,in which 5-HT signaling was involved.Experiment 3 The objective was to determine the effects of Trp on lipopolysaccharide(LPS)-induced glucose and amino acids transport in rats.Twenty-four 7-week Sprague Dawley rats were randomly divided into one of three groups(control,LPS or Trp + LPS).Mice were supplemented with 0 or 0.1 mg Trp/g BW/d in water for 7 d,and then were intraperitoneally injected with LPS(5 mg/kg BW)on d 8.After 24 h,rats were sacrificed to obain jejunal tissues for the analysis of transport of glucose and amino acids by the using of Ussing chamber.Results demonstrated that Trp alleviated LPS-induced elevation of intestinal permeability in the jejunum(P<0.05),and enhanced transport of glucose and amino acids(glutamate,arginine,glutamine,Trp,threonine and leucine)(P<0.05).LPS-induced down-regulation of glucose transporter(SGLT1)and animo acid transporters(SNAT2 and LAT2)as well as Na+/K+-ATPase(ATP1A2)were reversed by Trp(P<0.05).These findings demonstrated that dietary supplementation with Trp improved intestinal barrier function and attenuated gut inflammatory response by regulating tight junction proteins and inflammatory mediators,inhibiting inflammatory signaling pathways,and enhancing the transport of glucose and amino acids.5-HT signaling might be a critical molecule that conferred to the protective effect of Trp in intestinal inflammation.Trp may serve as effective nutritional stratege that can be used to prevent gut inflammation in animals and human beings.更多还原